Background/outline: Oncogenes and tumor suppressor genes (TSG) play a central role in causation and development of human cancer and provide the means to predict, detect, early diagnose and treat/cure human malignancies. The products of these cancer-causing genes are components of signal transduction circuits that control embryonic development, cellular proliferation and/or apoptotic death. Knowledge of these signaling pathways allows to identify molecular markers that herald the onset of cancer development and to predict response to treatment and clinical outcome. The development of tumors from incipient malignant cells to metastases is driven by Darwinian expansion of clonal cell populations involving additional mutated cancer genes. Every tumor tissue contains a minute population of tumor stem cells with self-renewal capacity essential for both tumor maintenance and spread. Fundamentally, these immortal tumor cells finally determine the response to treatment and tumor recurrence. Our research program began in 1987 and culminated in the identification in 1993 of the VHL TSG located at 3p25. In 1993-2003 we investigated the sequence structure of the VHL gene and identified and analyzed VHL target genes. In parallel we intensified research on mapping and molecular cloning of TSG located on 3p21.3 involved in the origin and /or development of major forms of lung cancer and other common carcinomas. In 2003-2004 we investigated: (1) the methylation code of the VHL locus itself and the function of VHL target genes (carbonic anhydrases, CA9 and CA12, the transcription regulator STRA13 and other VHL relay genes, STAT1 and TIEG1); (2) continued deletion mapping in 3p21.3 by real time PCR in tumor tissues and cell lines; and (3) completed the isolation and initial characterization of candidate cancer-causing genes from both 3p21.3 regions (centromeric, LUCA, and telomeric, AP20). These 3p21.3 regions should be considered contiguous cancer gene regions harboring clusters of TSG.We then focused our research on functional analysis (finding interacting proteins, analyzing null mutants in mice, and bioinformatics annotations) of some of the strong candidate genes.VHL TSG (3p25) Using the VHL locus as a model system, we investigated the mechanisms of aberrant DNA methylation in cancer. We also aimed to use epigenetic codes (CpG and H3/H4 histone codes) to understand whether they harbor variations associated with inter-individual/familial differences that may determine risk of sporadic VHL type cancers. We produced detailed and contiguous "bisulfite-sequencing" profiles of the human and mouse VHL loci (17 and 6.7 kb respectively) to reveal CpG codes in addition to H3/H4 histone methylation profiles. In VHL-expressing cells, only the promoter CpG island is protected and free from methylation while the rest of the locus is heavily methylated. This methylation pattern was re-created de novo and maintained on the human genomic VHL transgene during development of transgenic mice. Somatic cells were also able to preserve the pre-existing methylation pattern after fusion in somatic cell hybrids and after human-to-mouse single chromosome 3 transfer. We found that CTCF a ubiquitous chromatin DNA binding protein has binding sites in human and mouse VHL CpG promoter islands and may play an important role in protecting against aberrant silencing of the gene in kidney cancer. We will now focus on the identification of other protecting cis elements and binding proteins in the VHL locus and on studies of the VHL epigenetic code variations.We discovered that CA 9 /CA12 genes are specifically induced and over-expressed in many tumor types. These enzymes may control the acidic tumor microenvironment and should be considered molecular targets for development of new treatment modalities. Using purified CAIX/XII enzymes we tested novel and classical (clinically used for glaucoma treatment) aromatic sulfonamide inhibitors that may have potent anti-tumor activity. We identified among them several compounds that showed nanomolar inhibition specific for each enzyme. We aim now to initiate clinical trials with the FDA approved compounds. Our computer modeling studies of the enzyme-inhibitor interactions showed the affinity for each carbonic anhydrase correlates with how well the inhibitor fills the spatial volume of the inhibitor-binding pocket. This specificity is promising for the design of even more potent agents with reduced toxicity.The STRA13 transcription factor is negatively controlled by the VHL gene and induced by hypoxia, a condition prevalent in cancer tissues. We discovered that STRA13 binds to and modulates the activity of the powerful regulator of transcription STAT3 that is fundamentally involved in regulating cancer cell growth and survival and embryonic and probably cancer stem cell self-renewal. Importantly STRA13 is also involved in regulating acquired immunity. Our characterization of the STRA13 protein network is broadening the spectrum of potential therapeutic targets and expands our knowledge of the VHL carcinogenic pathway(s). Hypoxia and transforming growth factor -beta1 (TGF-beta 1) play important roles in regulation of the immune system, cell adhesion, migration, and tissue remodeling. Using TGF-beta1/BMP cDNA arrays and RNA isolated from CCRCC cell lines with different VHL status we outlined a set of targets common for pVHL and TGF-beta1 pathways. We associated one of these targets, the hypoxia-inducible Kruppel-like zinc finger transcriptional factor TIEG1, with transcriptional regulation of pVHL/hypoxia targets CA12, TGFBi, PAI-1, and others. We also showed that transcriptional modulation of another novel pVHL target, STAT1, is mediated via STRA13, which was previously implicated in both pVHL/HIF and TGF-beta1 pathways. These results provide novel insights on transcriptional cooperation between the pVHL/HIF, TGF-beta1, and JAK/STAT pathways and emphasize a new mechanism that employs relay genes to amplify and diversify the original primary hypoxia signal. In addition, these findings will provide a rapid and accurate identification of drug targets and tumor markers with true potential. The 3p21.3 TSG Functional studies on the 3p21.3 tumor suppressor genes: We used the yeast two-hybrid system, controlled expression of trangenes in tumor cells, targeted inactivation in mice (mouse knockout models), and bioinformatics to discover the function of the resident putative TSG.We identified the RASSF1A gene as a multiple TSG involved in many tumors, including lung, breast, prostate, kidney, head and neck, uterine cervix and others. We hypothesize that RASSF1 genes and their paralogs are inactivated in approximately 70% of human cancers.The HYAL2 protein was identified as a GPI-anchored receptor for the sheep lung cancer retrovirus, JSRV, and a sequestration mechanism inactivating HYAL2 protein was demonstrated. The env gene of JSRV was shown to transform human bronchial epithelial cells in vitro and sequester the HYAL2 protein. The absence of HYAL2 (mediated either by a putative virus or mutational inactivation) leads to ligand-independent activation of the RON receptor tyrosine kinase and its downstream signaling pathways (Akt and MAPK).We have been also studying the involvement of RON in SCLC. We discovered that in SCLC the promoter of RON is silenced by hypermethylation leading to simultaneous activation of a putative internal promoter. The novel transcript originating from this internal promoter encodes mostly the cytoplasmic portion of the receptor that is constitutively activated and may drive cell proliferation. Thus RON is emerging as a potential oncogenic factor in lung cancer and a target for therapeutic intervention.